1. Field of the Invention
Improving human and farm-animal nutrition, and reducing the environmental impact of agricultural production (as part of efforts to improve sustainability), are two highly ranked research goals. Cereal and legume-derived dietary phytic acid (myo-inositol 1,2,3,4,5,6-hexakisphosphate) impacts both areas.
Phytic acid is the major form of phosphorus (P) in seeds, typically representing from 65% to 80% of seed total P [Raboy, "Biochemistry and Genetics of Phytic Acid Synthesis". in Moore D. J., W. Boss, F. A. Loewus (eds). Inositol Metabolism in Plants, Alan R. Liss, New York. pp52-73 (1990)]. When seed-based diets are consumed by non-ruminants, the consumed phytic acid forms salts of several nutritionally important minerals in the intestinal tract. Excretion of these salts reduces the retention and utilization (ie. the bioavailability) of both their P and mineral contents. This phenomenon has been documented to contribute significantly to mineral deficiencies in both human and farm-animal populations [McCance et al., Biochem. J. 29:4269 (1935); Erdman, Cereal Chem. 58:21 (1981)]. Recently there has been growing concern over the contribution of animal-waste phytic acid P to surface and ground-water pollution [Jongbloed et al., Nether. J. Ag. Sci. 38:567 (1990)]. Kernel phytic acid P is also a problem in industrial uses such as wet milling, where it precipitates in the concentrated steep water, causing problems in handling, transportation and storage [Pen et al., Bio/tech. 11:811 (1993)].
Dietary phytic acid may also play positive roles, such as the inhibition of colonic cancer [Graf, Nutr. and Cancer 19:11 (1993)]. Thus, the relative merits or demerits of dietary phytic acid probably depends upon the population in question (ruminant vs. non-ruminant, human vs. farm animal) and the level of phytic acid in the staple diet. For example, in cases where the diet is high in phytic acid and the animal life span is short (such as in poultry and swine production), large reductions in dietary phytic acid may be desirable to improve mineral retention and reduce phosphate waste. In cases where life-span is long, but high dietary phytic acid is causing severe mineral deficiency, such as in numerous human populations in non-industrial societies, perhaps moderate reductions in dietary phytic acid would be desirable. In cases where life-span is long and dietary phytic acid is low, such as in certain industrial societies, increases in dietary phytic acid may be preferred.
This invention represents a novel approach to solving the aforementioned problems caused by phytic acid. A method is disclosed for isolating heritable low phytic acid (lpa) mutants of agronomically important seed crops. The mutants are the products of mutations which cause substantial reductions in kernel phytic acid but which have little effect on other plant or kernel characteristics, including kernel total P.
2. Description of the Prior Art
Several approaches to reducing the impact of dietary phytic acid on P and mineral retention, or reducing the problems caused by kernel phytic acid in industrial applications, such as wet-milling have been proposed or used. These approaches can be viewed as falling into two categories: 1) those which remove dietary phytic acid post-harvest; 2) those which attempt to reduce seed phytic acid content genetically, so that the initial product is improved and therefore does not require post-harvest technologies. The first category includes: food processing methods that remove phytic acid, either physically or via fermentation [Indumadhavi et al., Int. J. Food Sci. Tech. 27:221. (1992)]; and, more prominently, the use of phytases (phytic acid-specific phosphohydrolases typically of microbial origin) as dietary supplements [Nelson et al., J. Nutr. 101:1289 (1971)]. Pen et al. (WO91/14782) described transforming plants with constructs comprising a gene encoding phytase. Transgenic seed or plant tissues expressing phytases are then used as dietary supplements. Genetically reducing seed phytic acid was not mentioned as an objective of this technology and the effect of transformation with fungal phytases on seed phytic acid was not measured.
The second category involves developing crop germplasm possessing heritable reductions in seed phytic acid. Heritable, quantitative variation in seed phytic acid has been observed among lines of several crop species (reviewed in Raboy, supra). However, this variation has been found to be highly and positively correlated with variation in both plant and seed P, seed protein, and to a lesser extent seed mineral cation concentrations. Up to now, little or no heritable variation in these close and positive relationships has been reported. Therefore, breeding for reduced seed phytic acid using conventional breeding methods (some form of recurrent selection), would very likely result in germplasm having undesirable correlated characteristics. In any case, there have been no reports of "low phytic acid" or "phytic acid-free" germplasm, produced by such an approach, in use.
Recently it was proposed that a seed-specific block in phytic acid accumulation might be valuable in producing low phytic acid germplasm without introduction of undesirable correlated responses [Raboy et al., Crop Sci. 33:1300 (1993)] . At maturity the "wild-type" maize kernel typically contains about 3.5 mg/g phytic acid P, representing about 75% to 80% of kernel total P, and about 0.1 to 0.5 mg/g (0.2-1% total P) inorganic P. These values, and those of all phosphorus-containing fractions (total P, phytic acid P, inorganic P) throughout this disclosure are expressed as their phosphorus (P) content (MW 31), as opposed to their phosphate (PO.sub.4 H.sub.3) content (MW 98). The remaining kernel P is referred to as "cellular P". Phytic acid P usually represents greater than 95% of a mature maize kernel's water-extractable P. Phytic acid deposition is highly localized in the maize kernel. It accumulates in the aleurone layer (&lt;20%), and the germ (&gt;80%): ie. tissues peripheral to the endosperm. A survey of maize "defective kernel" (DEK) mutants, using the same "high-voltage paper electrophoresis" (HVPE) method described in the Examples below, revealed that those mutations which perturb germ and/or aleurone development often are associated with reductions in kernel phytic acid P, and that in all but one case these reductions are accompanied by equivalent increases in kernel inorganic P [Raboy et al., Maydica 35:383 (1990)]. The only exceptions are the viviparous (vp) mutants, in which precocious germination can also result in transient accumulations of the lower inositol phosphates (myo-inositol mono-, bis, tris, tetrakis, and pentakisphosphates). In all those cases, homozygosity for DEK mutants associated with substantial reductions in phytic acid proved to be lethal. Therefore, such mutants are not of any obvious agronomic value.